The present invention relates to a process for the production of a cross-linked polysaccharide gel with improved qualities and a gel obtainable by the process and use thereof. More precisely the invention refers to a new method of cross-linking, in which a bifunctional crossing-linking agent is introduced into the polysaccharide solution before emulsion and gel formation.
Chromatographic methods are commonly used for separation and purification of molecules such as proteins, nucleic acids, polysaccharides etc. A wide variety of separation media is available, both inorganic material as well as synthetic polymers and polysaccharides.
Gel matrices of polysaccharides have long been used as separation media due to their good qualities and such matrices are now standard equipment in biochemistry laboratories. The polysaccharides are inert to biomolecules under a wide range of conditions. The polysaccharides are natural materials and as such are approved of by authorities (such as the Food and Drug Administration (FDA)in USA) for many fields of application. When using chromatographic separation methods, there can be left traces of the separation medium left in the separated product. When polysaccharides are used, as separation medium, this is harmless, as the material is not toxic.
Generally, chromatographic separations are carried out in columns packed with the separation matrix in form of particulate beads. Separation media of a fast kinetics with rapid flow rates results in a high productivity and may be achieved by a reduction of the particle size. However, small beads result in a higher back pressure due to the narrowing of the convective flow channels between the particles in a packed bed. To be able to separate large molecules the particles should have large pores,but large pores may result in a weakened structure of the particles. As the polysaccharides are soft materials the particles may easily collapse, especially at high flow rates. Thus, there is a demand on methods of manufacturing more stable polysaccharide particles. It is well known to increase the stability of polysaccharide particles by cross-linking the polymer. The cross-linking process stabilises the polysaccharide gel matrices by chemically binding the polymer chains with each other at their respective free hydroxyl groups. The cross-linking takes place between the hydroxyl and the functional groups of the cross-linkers. This affects the particle rigidity, but to a lesser extent or not at all the size of the pores. There are several patents and articles disclosing different cross-linking methods. Well known cross-linking agents are epichlorohydrin, bis-epoxides, divinyl sulphone.
In EP 203 049 it was found that the rigidity of the polysaccharides was considerably improved when the cross-linking agent used was monofunctional but also contained an additional masked functional group that could be activated later. The cross-linking was made in two steps. First the polysaccharide was derivatized with the monofunctional group. Then, in a next step the masked group was activated and made to react with the hydroxyl groups of the polysaccharide. In this manner the length of the cross-linking was controlled and the desired rigidity obtained.
The common characteristic for the state of the art methods is that the cross-linking is made on the polysaccharide polymer after the formation of the gel particles. Thus, the cross-linking is made on the ready made structure. Particles of e.g. agarose are prepared by dissolving the agarose in water by heating. The hot water solution is then emulsified to form spherical particles in an organic solvent such as toluene. The particles are precipitated after cooling. The particles are then cross-linked. By varying the concentration of the agarose solution, different pore sizes can be obtained. The lower the concentration of the agarose solution the larger pores are obtained.
The object of the present invention was to obtain an improved process for the production of a cross-linked polysaccharide gel.
A further object of the invention was to produce rigid polysaccharide gel particles with improved capability to withstand high flow rates/back pressures, but with retained separation qualities.
The objects of the invention are achieved by the process and the polysaccharide gel as claimed in the claims. According to the invention a process for the production of a porous cross-linked polysaccharide gel is obtained, which process is characterized by the following steps:
a) preparing a solution or dispersion of the polysaccharide,
b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dispersion from step a),
c) reacting hydroxyl groups of the polysaccharide with the active site of the cross-linking agent,
d) forming a polysaccharide gel,
e) activating the inactive site of the cross-linking agent,
f) reacting the activated site from step e) with hydroxyl groups of the polysaccharide gel, whereby cross-linking of the gel takes place.
According to a further aspect of the invention a porous cross-linked polysaccharide gel is obtainable by the following steps:
a) preparing a solution or dispersion of the polysaccharide,
b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dispersion from step a),
c) reacting hydroxyl groups of the polysaccharide with the active site of the cross-linking agent,
d) forming a polysaccharide gel,
e) activating the inactive site of the cross-linking agent,
f) reacting the activated site from step e) with hydroxyl groups of the polysaccharide gel, whereby cross-linking of the gel takes place.
With the present invention it was surprisingly found that gels with increased pressure/flow capacities of more than 300% could be obtained, compared with known gels. It was possible to manufacture highly rigid gel particles also with small particle diameters (about 10 xcexcm).
According to the new method of the invention the cross-linking agent, is introduced into the polysaccharide solution or dispersion before the gel formation. The cross-linking agent is a bifunctional agent with one active site and one inactive site. When added to the polysaccharide solution or dispersion the active site of the agent is allowed to react with the hydroxyl groups of the polysaccharide. Thereby the cross-linking agent is chemically bound to the polymer chains before the gel formation process is started. In this manner an internal cross-linking agent is introduced into the polysaccharide.
In the first step of the process a solution or dispersion of the polysaccharide is formed. Solvents or dispersing agents commonly used together with polysaccharides can be used such as acetone, acetonitrile, dimethyl sulphoxide, dimethylformamide, pyridine, sec. and tert. alcohols, such as isopropanol, etc. However, according to a preferred embodiment of the invention an aqeous solution of the polysaccharide is used.
After the introduction of the cross-linking agent a gel is formed of the polysaccharide. If water has not been used as the solvent, the solvent or dispersing agent is then disposed of and the polysaccharide is dissolved in water. The gel is formed by emulsifying the water solution in an organic solvent such as toluene or heptane. Then, the inactive site of the cross-linking agent is activated and reacted with hydroxyl groups of the polysaccharide, whereby the gel is cross-linked.
The cross-linked gel can be further cross-linked by conventional methods as known by the state of the art. This further cross-linking can be made one or several times depending on how rigid particles that are required. The conventional cross-linking can also be made on the gel from step d) before or at the same time as activating and reacting of the inactive site of the cross-linking agent in steps e) and f). In a further embodiment of the invention steps b) and c) can be repeated one or several times after steps d) or e) in order to add more cross-linking agent before performing steps e) and f) or step f).
The bifunctional cross-linking agent used according to the invention comprises one active site and one inactive. With active site is meant all groups capable of reaction with the hydroxyl groups of the polysaccharide. Examples of such groups are halides, epoxides, methylol groups. The inactive site is a group which does not react under the reaction conditions for the reactive site but can later on be activated to react with the hydroxyl groups of the polysaccharide. Groups containing double bonds such as allyl, vinyl, acryl groups are suitable. The group connecting the active and inactive site is not essential, it should however, lack binding activity and not be too long. Preferable cross-linking agents are allylglycidyl ether and allylhalides, preferably allylbromide, but it is also possible to use e.g. N-methylol acrylamide, vinyl benzylchloride, cinnamoyl chloride. The reactions between the hydroxyl groups and the active site and the activated inactive site, as well as the activation of this site, is well known chemistry per see.
The reaction with the bifunctional cross-linking agent could be illustrated on agarose (AG) with the following reaction formulae:
Reaction with the active site of allylglycidyl ether: 
Activation and reaction of inactive site: 
The further cross-linking by conventional methods can be obtained by any of the known cross-linking agents. Suitable compounds are one or several from the group of epihalohydrin, bis-epoxides, divinylsulphone, allylglycidyl ether and dibromopropan-1-ol. Thus, the conventional cross-linking can be made with the same cross-linker as in the internal first cross-linking step or with another cross-linker or with a mixture of cross-linkers.
The process according to the invention can be used on all type of polysaccharides such as agarose, agarose derivatives, starch derivatives, dextran or derivatives thereof, alginate, carrageenan. However, agarose is the preferred one.
The gel matrix according to the invention is preferably prepared as particles. The manufacture of the gel is made with well known methods. Agarose for example, is dissolved in water by heating the solution above the gel point for agarose. The cross-linking agent is added to the hot aqueous agarose solution and the active site of the agent is allowed to react with the hydroxyl groups of the agarose. The agarose solution is then emulsified in an organic solvent such as toluene. The gel particles are precipitated by cooling. Thereafter, the inactive site of the cross-linking agent is activated and reacted with hydroxyl groups of the agarose particles, whereby the gel is cross-linked.
The size of the particles is determined by the stirring speed when emulsifying the agarose solution. The final required particle size is obtained by sieving. The pore sizes are regulated e.g. by varying the polysaccharide concentration. The process according to invention can be used to manufacture polysaccharide particles with conventional diameters and pore sizes. For the production of agarose particles the concentration suitably is from 0.5-20% by weight, preferably from 1-12% by weight. The particle diameters can be from 1 mm-1 xcexcm, preferably from 500 xcexcm -1 xcexcm, most preferably 200 xcexcm-1 xcexcm.
With the invention it is possible to produce highly rigid polysaccharide particles. The major parameter that influences the rigidity is the amount of added cross-linker, even if also the polysaccharide concentration has a significance for the rigidity and not only for the pore size as mentioned above. To obtain rigid particles the cross-linker concentration should preferably be within the range 30-80 xcexcmols/g gel, most preferably 45-60 xcexcmols/g gel. A concentration lower than 30 xcexcmols/g tends to give gels with relatively low pressure/flow capacities. A concentration above 80 xcexcmols/g can result in gels which shrink to much to be acceptable.
The porous cross-linked polysaccharide gel according to the invention can be used as a gel filtration medium in which the molecules are separated according to differences in their size. They can also be used, after modification, in different types of affinity chromatography. The gel can be substituted with a lot of different groups in per see known manners. Among such groups can be mentioned:
1. Positively charged groups (primary, secondary, tertiary or quaternary amino groups),
2. Negatively charged groups (e.g. carboxy, phosphonic acid, sulphonic acid etc.)
3. Amphoteric groups
4. Groups with specific affinity (e.g. such as between IgG-binding protein (protein A, G, L etc.) and IgG, lectin and carbohydrate, antigen/hapten and antibody, (strept)avidin and biotin,
5. Complementary nucleic acids/oligonucleotides,
6. Groups with pi-electron systems
7. Chelating groups
8. Hydrophobic groups.
With these groups the matrix can then be used in affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reversed phase chromatography, covalent chromatography etc.
The invention will now be illustrated by the following examples which however are not intended to limit the invention. With parts and percent are meant parts by weight and percent by weight if not stated explicitly.